Triterpenoid CDDO-EA inhibits lipopolysaccharide-induced inflammatory responses in skeletal muscle cells through suppression of NF-κB

Abstract

Chronic inflammation is a major contributor to the development of obesity-induced insulin resistance, which then can lead to the development of type 2 diabetes (T2D). Skeletal muscle plays a pivotal role in insulin-stimulated whole-body glucose disposal. Therefore, dysregulation of glucose metabolism by inflammation in skeletal muscle can adversely affect skeletal muscle insulin sensitivity and contribute to the pathogenesis of T2D. The mechanism underlying insulin resistance is not well known; however, macrophages are important initiators in the development of the chronic inflammatory state leading to insulin resistance. Skeletal muscle consists of resident macrophages which can be activated by lipopolysaccharide (LPS). These activated macrophages affect myocytes via a paracrine action of pro-inflammatory mediators resulting in secretion of myokines that contribute to inflammation and ultimately skeletal muscle insulin resistance. Therefore, knowing that synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acids (CDDOs) can attenuate macrophage pro-inflammatory responses in chronic disorders, such as cancer and obesity, and that macrophage pro-inflammatory responses can modulate skeletal muscle inflammation, we first examined whether CDDO-ethyl amide (CDDO-EA) inhibited chemokine and cytokine production in macrophages since this had not been reported for CDDO-EA. CDDO-EA blocked LPS-induced tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), interleukine-1beta (IL-1β), and interleukine-6 (IL-6) production in RAW 264.7 mouse and THP-1 human macrophages. Although many studies show that CDDOs have anti-inflammatory properties in several tissues and cells, little is known about the anti-inflammatory effects of CDDOs on skeletal muscle. We hypothesized that CDDO-EA protects skeletal muscle from LPS-induced inflammation by blocking nuclear factor kappa B (NF-κB) signaling. Our studies demonstrate that CDDO-EA prevented LPS-induced TNF-α and MCP-1 gene expression by inhibiting the NF-κB signaling pathway in L6-GLUT4myc rat myotubes. Our findings suggest that CDDO-EA suppresses LPS-induced inflammation in macrophages and myocytes and that CDDO-EA is a promising compound as a therapeutic agent for protecting skeletal muscle from inflammation.

Keywords: Inflammation; insulin resistance; macrophage; nuclear factor kappa B; skeletal muscle cells; triterpenoid.

Figure 1.

CDDO-EA inhibits LPS-induced cytokine secretion in macrophages. RAW264.7 mouse macrophages were pre-treated with 500 nM CDDO-EA for 1 h then exposed to 100 ng/mL LPS for 6 h. TNF-α levels were measured and quantified by ELISA. Three independent experiments were run in duplicate or triplicate. Data represented as mean ± SEM. *P < 0.05 (one-way ANOVA).

Figure 2.

CDDO-EA blocks LPS-induced IL-1β, IL-6, and MCP-1 secretion in THP-1 macrophages. THP-1 macrophages were pre-treated with 500 nM CDDO-EA for 1 h then exposed to 100 ng/mL LPS for 6 h. Cytokine and chemokine levels were measured and quantified by a Bio-Plex Pro-Human 17-plex ELISA kit. Experiment was run in triplicate. Data represented as mean ± SEM. **P < 0.01; ***P < 0.001; (one-way ANOVA). NS: not significant.

Figure 3.

CDDO-EA inhibits LPS-induced NF-κB and IκB phosphorylation: (A) L6-GLUT4myc myotubes were pre-treated with 500 nM CDDO-EA for 1 h then exposed to 100 ng/mL LPS for 1 h. Myotubes were harvested for analysis by Western blot using antibodies against phosphorylated NF-κB p65 (Ser536), total NF-κB, phosphorylated IκB-α (Ser32), IκB-α total protein, and actin. NF-κB and IκB-α phosphorylation was normalized to NF-κB and IκB-α total protein, respectively, then expressed as percent over control sample. Four independent experiments were run in duplicate or triplicate. (B) L6-GLUT4myc myotubes were treated with doubling doses of CDDO-EA for 6 or 24 h. Cell viability was normalized to non-treated myotubes. Two independent experiments were run in triplicate. Data are represented as mean ± SEM. *P < 0.05; ***P < 0.001; (one-way ANOVA).

Figure 4.

CDDO-EA affects NF-κB translocation. L6-GLUT4myc myotubes were pre-treated with 500 nM CDDO-EA for 1 h followed by 100 ng/mL LPS for 1 h. (A) For immunofluorescence, fixed, and permeabilized myotubes were stained using DAPI (nuclei, blue) and anti-NF-κB p65 primary antibody and a secondary antibody conjugated with Alexa Fluor 488. NF-κB was presented with green fluorescence in cytoplasm and nuclei (red arrows). Scale bar: 50 µm. Images are representative of two independent experiments. (B) Cytoplasmic and nuclear fractions were isolated and immunoblotted for p65 NF-κB and GAPDH and lamin to verify cytoplasmic and nuclear fractions, respectively. Data are representative of three independent experiments run in duplicate.

Figure 5.

CDDO-EA blocks NF-κB transcriptional activity. (A) L6-GLUT4myc myotubes were transfected with an NF-κB luciferase reporter plasmid then treated with 500 nM CDDO-EA for 1 h followed by 100 ng/mL LPS for 6 h. Data indicate luciferase values normalized to total protein concentration. Experiment for each time point was run in duplicate. (B) GLUT4myc myotubes were exposed to 500 nM CDDO-EA for 1 h followed by 1 h exposure with 100 ng/mL LPS. mRNA expression levels of TNF-α and MCP-1 were measured by RT-PCR. Data are representative of two independent experiments run in triplicate (mean ± SEM). *P < 0.05; **P < 0.01 ***P < 0.001; (one-way ANOVA); RLU, relative light unit.

Figure 6.

CDDO-EA activates GLUT4 translocation to cell membrane and p38 phosphorylation. L6-GLUT4myc myotubes were pre-treated with 500 nM CDDO-EA for 1 h followed by 100 ng/mL LPS for 1 h. 100 nM insulin treatment was for 15 min. (A) For immunofluorescence, myotubes were fixed without permeabilization and stained using DAPI (nuclei, blue) and anti-c-myc primary antibody (c-myc epitope-tagged GLUT4 distinguishes from endogenous GLUT4) and a secondary antibody conjugated with Alexa Fluor 488. c-myc was presented with green fluorescence. Scale bar: 50 µm. (B) Myotubes were harvested for analysis by Western blot using antibodies against phosphorylated p38, total p38, and actin. p38 phosphorylation was normalized to p38 total protein, then expressed as percent over control sample. Three independent experiments were run in triplicate. Data are represented as mean ± SEM. ***P < 0.001; (unpaired t-test, two-tailed).